Therapeutic particles suitable for parenteral administration and methods of making and using same

ABSTRACT

Disclosed herein are therapeutic compositions for treating and preventing diseases such as neointimal hyperplasia (NIH), where the compositions comprise a therapeutic particle that has a localized association with a blood vessel and a therapeutic agent, such as an anti-NIH agent. Methods of use of the therapeutic compositions are also disclosed.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/636,105, filed Dec. 11, 2009, which claims priority to U.S. Ser. No.61/122,046, filed Dec. 12, 2008, U.S. Ser. No. 61/159,625, filed Mar.12, 2009, and U.S. Ser. No. 61/240,433 filed Sep. 8, 2009, each of whichis hereby incorporated by reference in its entirety.

BACKGROUND

The process of restonosis, or renarrowing of an e.g. a coronary arterylumen following a revascularization procedure, most likely begins at thetime of percutaneous intervention. Restenosis typically can involvemechanical processes such as elastic recoil, or acute renarrowing of anartery after e.g. balloon angioplasty, and processes such as negativearterial remodeling—as the vessel begins to heat, the outermost vessellayer (the adventitial layer) may shrink inward. Neointimal hyperplasia(NIH), another process in the development of restenosis, is not amechanical function of the anatomy of an artery, but a biological woundhealing response to the injury caused by percutaneous coronaryintervention. Neointimal hyperplasia can involve smooth muscle cellproliferation, migrations and/or production of the extracellular matrix.With the introduction of arterial stents, the problems of elastic recoilor negative arterial remodeling has been substantially eliminated.However, neointimal hyperplasia is still a primary cause of restenosisafter the introduction of a stent in the artery of a patient. Both baremetal stents and drug-eluting stents that typically elute ananti-restenosis agent are available.

Drug eluting stents are not optimal under all conditions, however. Suchstents may not be appropriate for small vessels, and drug eluting stentscan hinder vessel healing. For example, drug eluting stents may lead toa higher rate of thrombosis e.g. a year after implantation, when forexample antiplatelet therapy (e.g. clopidogrel) is discontinued. Forpatients needing surgery, for example, patients may suffer fatal heartattacks due to clotting inside of drug-eluting stents, even months oryears after surgery, particular if blood thinning medication is stopped(as is often necessary) before surgery. Patients identified as beinglikely to be non-compliant with antiplatelet therapy may not be suitablecandidates for drug-eluting stents.

Therefore, it has been suggested that use of bare metal stents mayactually provide a safer choice, at least for some patients. Improvedcompositions and methods for delivery of anti-neointimal hyperplasia, oranti-restenosis, agents for local and/or targeted delivery to bloodvessels, for example, in conjunction with placement of a bare-metalstents is therefore needed.

SUMMARY

This disclosure is generally directed to therapeutic compositions thatinclude therapeutic particles comprising an anti-neointimal hyperplasia(NIH) agent. Such therapeutic particles may be capable of releasing saidanti-NIH agent to a blood vessel, for example, to a basement vascularmembrane of a blood vessel. For example, such particles may be capableof releasing an anti-NIH agent for at least about 8 hours when adisclosed therapeutic particle or composition is placed in the bloodvessel. Such compositions may be used, for example, with a patientreceiving a vascular stent, e.g. a bare metal stent, in a blood vessel.

For example, the disclosure provides a therapeutic compositioncomprising a plurality of therapeutic particles each comprising ananti-NIH agent and a basement vascular membrane targeting peptide,wherein a single administration of a dose of the composition to a bloodvessel is capable of contacting a blood vessel with a substantiallyhigher surface area density as compared to a surface area density of ablood vessel contacted by a stent comprising the anti-NIH agent.

In an embodiment, the disclosure provides a method of preventing ordeterring NIH in a blood vessel of a patient receiving a bare metalstent in a lesion of said blood vessel, comprising administering acomposition comprising therapeutic particles, wherein said therapeuticparticles comprise a basement vascular membrane targeting peptide and ananti-NIH agent. In some embodiments, methods disclosed herein includeintraveneous (e.g., systemic) administration of disclosed compositionsand/or particles.

Also provided herein, in an embodiment, is a method of preventing ordeterring NIH in a damaged blood vessel of a patient, comprisingadministering to said patient a therapeutic composition comprising atherapeutic particle, wherein said therapeutic particle may comprise abasement vascular membrane targeting peptide and an anti-NIH agent, andwherein said therapeutic particle substantially biodegrades afterdelivery of the anti-NIH agent thereby promoting healing of the bloodvessel after said therapeutic particle has biodegraded.

Therapeutic compositions are provided herein, for example, for use in apatient receiving a vascular stent, comprising about 1 to about 20 molepercent targeting co-polymer wherein the targeting co-polymer is chosenfrom: a) PLA-PEG-basement vascular membrane targeting peptide; b) poly(lactic) acid—co poly (glycolic) acid-PEG-basement vascular membranetargeting peptide; c) DSPE-PEG-basement vascular membrane targetingpeptide; and about 0.2 to about 30 weight percent anti-neointimalhyperplasia (NIH) agent (e.g. paclitaxel); about 50 to about 90 weightpercent non-targeted polylactic acid or poly-lactic acid-PEG. Forexample, such a targeting co-polymer may include PLA-PEG, wherein forexample poly(lactic acid) has a number average molecular weight of about15 to 20 kDa and/or poly(ethylene)glycol has a number average molecularweight of about 4 to about 6 kDa. Contemplated basement vascularmembrane targeting peptides may be selected, for example, from: CREKA(SEQ ID NO: 1) or CARLYQKLN (SEQ ID NO: 2).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts three methods for delivery of compositions of theinvention to a target site in a blood vessel, where the blood flow 20 isfrom left to right in the diagram. FIG. 1A illustrates delivery by guidecathether in the blood flow. FIG. 1B illustrates use of a ballooncatheter for proximal delivery. FIG. 1C illustrates the use of a guidecatheter with a balloon catheter for distal delivery.

FIG. 2 depicts the effect of lipid content and number of homogenizerpasses on particle size using DSPE-PEG5K.

FIG. 3 depicts the effect of lipid content and number of homogenizerpasses on particle size using DSPE-PEG5K.

FIG. 4 depicts release of paclitaxel loaded particles at 37° C.

FIG. 5 depicts release of various paclitaxel loaded particles at 37° C.

FIG. 6 depicts release of various paclitaxel loaded particles.

FIG. 7 depicts pharmacokinetics of various pacitaxel loaded particles ina rabbit model.

FIG. 8 depicts the paclitaxel release rate of disclosed nanoparticleswith paclitaxel, in a rabbit model.

FIG. 9 depicts the paclitaxel blood levels in a disclosed rabbit model.

FIG. 10 depicts injured artery paclitaxel concentration in a rabbitmodel, after administration of disclosed nanoparticles havingpaclitaxel.

DETAILED DESCRIPTION

The features and other details of the disclosure will now be moreparticularly described. Before further description of the presentinvention, certain terms employed in the specification, examples andappended claims are collected here. These definitions should be read inlight of the remainder of the disclosure and understood as by a personof skill in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by a person of ordinary skill in the art.

DEFINITIONS

“Treating” includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder and the like.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, coatings, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions.

“Individual,” “patient,” or “subject” are used interchangeably andinclude to any animal, including mammals, such as mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans. The compounds and compositions of theinvention can be administered to a mammal, such as a human, but can alsobe other mammals such as an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like). The mammal treated in themethods of the invention is desirably a mammal in whom modulation of NIHis desired. “Modulation” includes antagonism (e.g., inhibition),agonism, partial antagonism and/or partial agonism.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound or composition thatwill elicit the biological or medical response of a tissue, system,animal or human that is being sought by the researcher, veterinarian,medical doctor or other clinician. The compounds and compositions of theinvention are administered in therapeutically effective amounts to treata disease. Alternatively, a therapeutically effective amount of acompound is the quantity required to achieve a desired therapeuticand/or prophylactic effect, such as an amount which results in theprevention of or a decrease in the symptoms associated with NIH.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that include an aminomoiety may form pharmaceutically acceptable salts with various aminoacids, in addition to the acids mentioned above. Compounds included inthe present compositions that are acidic in nature are capable offorming base salts with various pharmacologically acceptable cations.Examples of such salts include alkali metal or alkaline earth metalsalts, such as calcium, magnesium, sodium, lithium, zinc, potassium, andiron salts.

This disclosure provides, at least in part, therapeutic compositionsthat include a therapeutic particle. Such therapeutic particles caninclude for example an anti-neointimal hyperplasia (NIH) agent orantiflammatory agent, and may be capable of releasing the anti-NIH agentto a vascular membrane of a blood vessel for at least about 2, 4, 6, 8,10, 12, or even 24 or more hours, or 1, 2 or 3 or more days, or forabout 1, 2, 4 or even 12 or more weeks, when the therapeutic particle isplaced in the blood vessel. In some embodiments, a disclosed therapeuticparticle may include basement vascular membrane targeting peptide.

In some embodiments, disclosed therapeutic compositions may be for use,e.g., administered to or with a patient receiving a stent such as avascular stent, for example, a bare metal stent, or, in someembodiments, a drug-eluting stent. In an embodiment, disclosedcompositions may release substantially equal or substantially moreeffective amount of the anti-NIH agent when placed in the blood vesselas compared to the amount released by a stent comprising the sameanti-NIH agent if placed in the blood vessel. In some embodiments, adisclosed therapeutic composition may release about 10% to about 50% ormore, e.g., about 40%, or about 30% of the anti-NIH agent initially,with a more controlled release of the remaining drug over time.

In one embodiment, the anti-NIH agent can be chosen from paclitaxel,sirolimus, zotarolimus and/or everolimus. For example, a disclosedtherapeutic particle may comprise paclitaxel such that the therapeuticcomposition releases about 50 μg to about 1000 μg, about 50 μg to about600 μg, or about 100 μg to about 300 μg, of paclitaxel to a target ofinterest, e.g. the basement vascular membrane. Such release can occurover a period of about 8 hours to about 8 weeks, such as about 8 hoursto about 4 weeks, further such as about 8 hours to about 1 week. Inanother exemplary embodiment, a disclosed therapeutic particle maycomprise sirolimus such that the therapeutic composition releases about50 μg to about 250 μg, such as 50 μg to about 100 μg, about 75 μg toabout 150 μg, of sirolimus to, e.g., the basement vascular membrane. Atherapeutic particle may comprise zotarolimus, for example, such thatthe therapeutic composition releases about 50 μg to about 300 μg, suchas about 50 μg to about 250 μg, or about 75 μg to about 150 μg, ofzotarolimus to e.g., a basement vascular membrane. In anotherembodiment, the therapeutic particle may comprise everolimus, forexample, such that the therapeutic composition releases about 50 μg toabout 300 μg, such as about 50 μg to about 250 μg, further such as about75 μg to about 150 μg, of everolimus to the basement vascular membrane.The release of such drugs can occur over a period of about 8 hours toabout 8 weeks, such as about 8 hours to about 4 weeks, further such asabout 8 hours to about 1 week.

Therapeutic particles disclosed herein may include about 5% to about 85%by weight of the anti-NIH agent, such as about 2% to about 35%, or about10% to about 25%, e.g. about 10%, 15%, or 20% by weight. The therapeuticparticle may be substantially biodegraded after about 1 month, afterabout 1 week, such as about 3 days, further such as about 1 day, afterplacement in the blood vessel.

In some embodiments, disclosed compositions may provide a decreased orsubstantially comparable NIH rate in the patient about 4 weeks, or 3weeks, or 2 weeks, such as about 1 week, after receiving the vascularstent as compared to the NIH rate obtained by administration of avascular stent alone. In another embodiment, the NIH rate may bedecreased compared to that from administration of a vascular stent, e.g.a bare metal stent, alone.

Also provided herein are therapeutic compositions that include aplurality of therapeutic particles each comprising an anti-NIH agent andoptionally, a basement vascular membrane targeting peptide, wherein asingle administration of a dose of the composition to a blood vessel canbe capable of contacting a blood vessel with a substantially highersurface area density as compared to a surface area density of a bloodvessel, e.g a surface area density of endothelial cells, as compared tothe surface area density contacted by a stent comprising the anti-NIHagent. In some embodiments, one or more doses of the composition, whenadministered, may contact the blood vessel with a substantially highersurface area density as compared to the density of blood vesselcontacted by a stent that includes the anti-NIH agent. For example,compositions may contact least a surface area density of about leastabout 2%, such as at least 5%, such as at least 10% higher than thedensity of blood vessel contacted by a stent comprising the same ordifferent anti-NIH agent, e.g. a drug-eluting stent.

Doses of disclosed compositions may be capable of delivering theanti-NIH agent to the blood vessel such that the concentration of theanti-NIH agent in the blood vessel tissue can be about 2 ng/mg to about100 ng/mg, such as about 15 ng/mg to 50 ng/mg, further such as about 20ng/mg to 40 ng/mg about 2 days after administration.

Another embodiment provides therapeutic compositions, e.g., for use in apatient receiving a vascular stent, comprising a plurality of firsttherapeutic particles, wherein the first therapeutic particles may becapable of localized association with a blood vessel structure, e.g. abasement membrane, smooth muscle cells, endothelial cells, extracellularmatrix, or inner elastic lamina, and comprise a first therapeutic agent,e.g. an anti-NIH agent, and wherein a single administration of a dose ofthe composition to the blood vessel provides a faster endothelial cellhealing rate at about 6 months, 4 months, or about 2 months, as comparedto the endothelial healing rate of a patient receiving a stentcomprising the first therapeutic agent at about 6 months. In anotherembodiment, a single administration (or multiple administrations) of adose of a composition associates with a greater surface area density ofthe blood vessel structure as compared to a patient receiving a stentcomprising the first therapeutic agent. Such compositions may furtherinclude a plurality of second therapeutic particles. The secondtherapeutic particles may be capable of localized association with adifferent blood vessel structure than the first therapeutic particles.The second therapeutic particles may comprise a second therapeuticagent, which may be different than the first therapeutic agent.

Also provided herein are therapeutic compositions that include aplurality of first therapeutic particles, wherein the first therapeuticparticles may be capable of localized association with a blood vesselstructure and comprise a first therapeutic agent such as an anti-NIHagent or anti-inflammatory agent; and a plurality of second therapeuticparticles comprising a second therapeutic agent. In one embodiment, theplurality of first therapeutic particles can be present in a differentamount than the plurality of second therapeutic particles. In anotherembodiment, the composition can comprise an equal amount of first andsecond therapeutic particles. A target of the first therapeutic agentmay be different than a target of the second therapeutic agent. Suchcompositions may release a substantially equal or substantially greateramount of first therapeutic agent than second therapeutic agent, e.g.another anti-NIH agent or anti-inflammatory, in the blood vessel.

Exemplary second therapeutic agents include, but are not limited to,everolimus, paclitaxel, zotarolimus, pioglitazone, BO-653,rosiglitazone, sirolimus, dexamethasone, rapamycin, tacrolimus,biophosphonates, estrogen, angiopeptin, statin, PDGF inhibitors, ROCKinhibitors, MMP inhibitors, 2-CdA, corticosteroids, includingcombinations of zotarolimus and dexamethasone, nicotine,hydroxy-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors,statins, niacin, bile acid resins, fibrates, antioxidants, nitric oxidegenerators, nitric oxide, extracellular matrix synthesis promoters,inhibitors of plaque inflammation, and extracellular degradation, andantithrombotic agents suchs as clopidogrel.

Contemplated anti-inflammatories or anti-inflammatory agents, which maybe useful for restenosis, (e.g. vessel remodeling that occurs inrestenosis starts with inflammation in response to the injury caused byangioplasty, and stopping this inflammation has been showed in animalstudies to prevent restenosis), include sirolimus, corticosteroids(dexamethasone, prednisolone, triamcinolone acetonide, mometasone,amcinonide, budesonide, acetaminophen, NSAIDS, cox-2 inhibitors, and/orbetamethasone.

Methods contemplated herein include, for example, a method of preventingor deterring NIH in a blood vessel of a patient receiving a bare metalstent in a lesion of the blood vessel, comprising administering acomposition comprising disclosed therapeutic particles, such astherapeutic particles that may include a basement vascular membranetargeting peptide and an anti-NIH agent or anti-inflammatory. Disclosedmethods may provides for decreased or substantially comparable NIH rateat about 2 weeks, such as about 1 week, after receiving the bare metalstent as compared to a patient receiving a stent comprising an anti-NIHagent at about 2 weeks or at about 1 week. In some embodiments, methodsare provided that include administering or placing a bare metal stent ina blood vessel, and administering a disclosed therapeutic particle orcomposition before, after, or substantially simultaneously with theplacement of the stent. The blood vessel receiving the stent may be lessthan about 2 mm in length, or between about 2 mm and about 3 mm inlength, or may be greater than 3 mm in length. A treated blood vesselmay be bifurcated or substantially non-bifurcated.

Such stents for use in the contemplated methods may be a thick or thinstrut stent. For example, stents may be less than about 14 mm, such asless than about 10 mm, further such as less than about 7 mm in length.In another embodiment, contemplated stents may be about 14 mm to about30 mm, such as about 17 mm to about 27 mm, further such as about 20 mmto about 25 mm in length.

In some embodiments, a patient being treated or contemplating treatment,may be intolerant or adverse to a particular medication, such as aspirinor clopidogrel. Patient populations suitable for treatment withdisclosed methods include patients at risk of future surgery, e.g.,cardiac or non-cardiac surgery.

Contemplated compositions or particles may be administered substantiallysimultaneously when a patient receives the stent and/or may beadministered before or after the patient receives the stent. Forexample, compositions and/or particles may be introduced before or afterthe introduction of a balloon catheter into the blood vessel. Thecomposition may be administered with the same delivery device used todeliver the stent to the patient, or a different delivery device. In oneembodiment, the composition may be administered using a catheter, and/ormay be administered intravenously. Disclosed compositions may beadministered to a patient undergoing, for example, a coronaryangioplasty, a peripheral angioplasty, a renal artery angioplasty, or acarotid angioplasty.

Another embodiment provides a method of preventing or deterring NIH in adamaged blood vessel of a patient, comprising administering to thepatient a therapeutic composition comprising a therapeutic particle,wherein the therapeutic particle comprises a basement vascular membranetargeting peptide and an anti-NIH agent, and wherein the therapeuticparticle substantially biodegrades after delivery of the anti-NIH agentthereby promoting healing of the blood vessel after the therapeuticparticle has biodegraded. For example, a therapeutic particle may havesubstantially degraded about 1 day, such as about 1 week, further suchas about 1 month after administration. The damaged blood vessel may havebeen caused by, for example, an implantation of a stent (e.g., a baremetal stent), a balloon angioplasty, or peripheral artery disease. In anexemplary embodiment, a damaged blood vessel may be caused, for example,by balloon angioplasty alone. Such methods may be suitable for patientswhere placement or administration of a stent (e.g. a bare metal stent ora drug eluting stent) is not appropriate, for example, patientssuffering from superficial femoropopliteal artery obstructions orocclusions. After administration of disclosed particles or compositions,a blood vessel, may, in some embodiments, have substantially less riskof developing a thrombosis at about 1 month, or greater than about 1month, about 2 months, further such as greater than about 3 months afteradministration as compared to a damaged blood vessel receiving a stentcomprising an anti-NIH agent, e.g., a drug-eluting stent.

Therapeutic particles disclosed herein typically include a polymericmatrix. In one embodiment, the polymeric matrix comprises one, two ormore synthetic or natural polymers. The term “polymer,” as used herein,is given its ordinary meaning as used in the art, i.e., a molecularstructure comprising one or more repeat units (monomers), connected bycovalent bonds. The repeat units may all be identical, or in some cases,there may be more than one type of repeat unit present within thepolymer. In some cases, the polymer can be biologically derived, i.e., abiopolymer. Non-limiting examples include peptides or proteins. In somecases, additional moieties may also be present in the polymer, forexample biological moieties such as those described below. If more thanone type of repeat unit is present within the polymer, then the polymeris said to be a “copolymer.” It is to be understood that in anyembodiment employing a polymer, the polymer being employed may be acopolymer in some cases. The repeat units forming the copolymer may bearranged in any fashion. For example, the repeat units may be arrangedin a random order, in an alternating order, or as a block copolymer,i.e., comprising one or more regions each comprising a first repeat unit(e.g., a first block), and one or more regions each comprising a secondrepeat unit (e.g., a second block), etc. Block copolymers may have two(a diblock copolymer), three (a triblock copolymer), or more numbers ofdistinct blocks.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, the polymer (e.g., copolymer, e.g., blockcopolymer) can be amphiphilic, i.e., having a hydrophilic portion and ahydrophobic portion, or a relatively hydrophilic portion and arelatively hydrophobic portion. A hydrophilic polymer can be onegenerally that attracts water and a hydrophobic polymer can be one thatgenerally repels water. A hydrophilic or a hydrophobic polymer can beidentified, for example, by preparing a sample of the polymer andmeasuring its contact angle with water (typically, the polymer will havea contact angle of less than 60°, while a hydrophobic polymer will havea contact angle of greater than about 60°). In some cases, thehydrophilicity of two or more polymers may be measured relative to eachother, i.e., a first polymer may be more hydrophilic than a secondpolymer. For instance, the first polymer may have a smaller contactangle than the second polymer.

In one set of embodiments, a polymer (e.g., copolymer, e.g., blockcopolymer) contemplated herein includes a biocompatible polymer, i.e.,the polymer that does not typically induce an adverse response wheninserted or injected into a living subject, for example, withoutsignificant inflammation and/or acute rejection of the polymer by theimmune system, for instance, via a T-cell response. Accordingly, thetherapeutic particles contemplated herein can be non-immunogenic. Theterm non-immunogenic as used herein refers to endogenous growth factorin its native state which normally elicits no, or only minimal levelsof, circulating antibodies, T-cells, or reactive immune cells, and whichnormally does not elicit in the individual an immune response againstitself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEGylated polymers andcopolymers of lactide and glycolide (e.g., PEGylated PLA, PEGylated PGA,PEGylated PLGA, and derivatives thereof. In some embodiments, polyestersinclude, for example, polyanhydrides, poly(ortho ester) PEGylatedpoly(ortho ester), poly(caprolactone), PEGylated poly(caprolactone),polylysine, PEGylated polylysine, poly(ethylene imine), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA can be characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid-glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention can be characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, the ratio of lactic acid to glycolic acid monomersin the polymer of the particle (e.g., the PLGA block copolymer orPLGA-PEG block copolymer), may be selected to optimize for variousparameters such as water uptake, therapeutic agent release and/orpolymer degradation kinetics can be optimized.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine), polyethylene imine(PEI), and poly(amidoamine) dendrimers are contemplated for use, in someembodiments, in a disclosed particle.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains. Examples of these polyesters includepoly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester). A polymer (e.g., copolymer, e.g., blockcopolymer) containing poly(ethylene glycol) repeat units can also bereferred to as a “PEGylated” polymer. Such polymers can controlinflammation and/or immunogenicity (i.e., the ability to provoke animmune response) and/or lower the rate of clearance from the circulatorysystem via the reticuloendothelial system (RES), due to the presence ofthe poly(ethylene glycol) groups.

PEGylation may also be used, in some cases, to decrease chargeinteraction between a polymer and a biological moiety, e.g., by creatinga hydrophilic layer on the surface of the polymer, which may shield thepolymer from interacting with the biological moiety. In some cases, theaddition of poly(ethylene glycol) repeat units may increase plasmahalf-life of the polymer (e.g., copolymer, e.g., block copolymer), forinstance, by decreasing the uptake of the polymer by the phagocyticsystem while decreasing transfection/uptake efficiency by cells. Thoseof ordinary skill in the art will know of methods and techniques forPEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminating inan amine, by ring opening polymerization techniques (ROMP), or the like.

Particles disclosed herein may or may not contain PEG. In addition,certain embodiments can be directed towards copolymers containingpoly(ester-ether)s, e.g., polymers having repeat units joined by esterbonds (e.g., R—C(O)—O—R′ bonds) and ether bonds (e.g., R—O—R′ bonds). Insome embodiments of the invention, a biodegradable polymer, such as ahydrolyzable polymer, containing carboxylic acid groups, may beconjugated with poly(ethylene glycol) repeat units to form apoly(ester-ether).

In one embodiment, the molecular weight of the polymers can be optimizedfor effective treatment as disclosed herein. For example, the molecularweight of a polymer may influence particle degradation rate (such aswhen the molecular weight of a biodegradable polymer can be adjusted),solubility, water uptake, and drug release kinetics. For example, themolecular weight of the polymer can be adjusted such that the particlebiodegrades in the subject being treated within a reasonable period oftime (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8weeks, etc.). A disclosed particle can for example comprise a copolymerof PEG and PLGA, the PEG can have a molecular weight of 1,000-20,000,e.g., 5,000-20,000, e.g., 10,000-20,000, and the PLGA can have amolecular weight of 5,000-100,000, e.g., 20,000-70,000, e.g.,20,000-50,000.

In some embodiments, disclosed therapeutic particles and/or compositionsinclude targeting agents such as dyes, for example Evans blue dye. Suchdyes may be bound to or associated with a therapeutic particle, ordisclosed compositions may include such dyes. For example, Evans bluedye may be used, which may bind or associate with albumin, e.g. plasmaalbumin.

Disclosed therapeutic particles, may, some embodiments, include atargeting moiety, i.e., a moiety able to bind to or otherwise associatewith a biological entity, for example, a membrane component, a cellsurface receptor, Her-2, the basement membrane of a blood vessel,basement membrane proteins, collagen, collagen IV or the like. Forexample, the targeting moiety can be a basement vascular membranetargeting peptide. The term “bind” or “binding,” as used herein, refersto the interaction between a corresponding pair of molecules or portionsthereof that exhibit mutual affinity or binding capacity, typically dueto specific or non-specific binding or interaction, including, but notlimited to, biochemical, physiological, and/or chemical interactions.disassociation constant).

For example, a targeting moiety may target tissue basement membrane,such as the basement membrane of a blood vessel. A “basement membrane”refers to a thin membrane upon which is posed about a single layer ofcells. For example, a basement membrane can be made up of proteins heldtogether by type IV collagen. Epithelial cells are anchored withhemidesmosome to the basement membrane. The end result resembles a layerof tiles attached to a thin sheet. In cases where the endothelium can bedisrupted (by disease or trauma, e.g. the process of stent placement),the basement membrane may be exposed and accessible to particles.

In one embodiment, the targeting peptide included in a particle may havea length of at most 200 residues. In another embodiment, the targetingpeptide or peptidomimetic portion of the particle can have a length ofat most 50 residues. For example, a disclosed particle may include atargeting peptide or peptidomimetic that includes the amino acidsequence AKERC (SEQ ID NO: 3), CREKA (SEQ ID NO: 1), ARYLQKLN (SEQ IDNO: 4), CARYLQKLN (SEQ ID NO: 2) or AXYLZZLN (SEQ ID NO: 5), wherein Xand Z can be variable amino acids, or conservative variants orpeptidomimetics thereof. In some embodiments, the poly(amino acid)targeting moiety can be a peptide that includes the amino acid sequenceAKERC (SEQ ID NO: 3), CREKA (SEQ ID NO: 1), ARYLQKLN (SEQ ID NO: 4) orAXYLZZLN (SEQ ID NO: 5), wherein X and Z can be variable amino acids,and can have a length of less than 20, 50 or 100 residues. Any peptide,or conservative variants or peptidomimetics thereof, that binds or formsa complex with collagen IV, or the basement membrane of a blood vessel,is contemplated for use as a targeting moiety.

In one embodiment, the targeting moiety can be an isolated peptide orpeptidomimetic that can have a length of less than 100 residues andincludes the amino acid sequence CREKA (Cys Arg Glu Lys Ala) (SEQ IDNO: 1) or a peptidomimetic thereof. Such an isolated peptide orpeptidomimetic can have, for example, a length of less than 50 residuesor a length of less than 20 residues. In some embodiments, the inventionprovides a peptide that includes the amino acid sequence CREKA (SEQ IDNO: 1) and can have a length of less than 20, 50 or 100 residues.

An exemplary embodiment includes a particle having a portion of thepolymer matrix covalently bound to a peptide, such as the basementvascular membrane targeting peptide—e.g., a peptide may form a ligand onthe polymer. Such covalent association may be through a linker, apolymer matrix can be covalently bound to the peptide via the freeterminus of e.g., a PEG or e.g., can be covalently bound to the peptidevia a carboxyl group at the free terminus of PEG. In another embodiment,the polymer matrix can be covalently bound to the peptide via amaleimide functional group at the free terminus of PEG.

In one embodiment, the ratio of peptide-bound polymer to free polymercan be selected to optimize the delivery and/or release of the anti-NIHagent to the basement vascular membrane of the blood vessel, or healingrate of the endothelieal cells. For example, increased ligand density(e.g., on a PLGA-PEG copolymer) may increase target binding (cellbinding/target uptake). Alternatively, a certain concentration ofnonfunctionalized polymer (e.g., non functionalized PLGA-PEG copolymer)in the therapeutic particle may control inflammation and/orimmunogenicity (i.e., the ability to provoke an immune response), mayallow a particle to have a circulation half-life that can betherapeutically effective for the treatment of NIH. Furthermore, anon-functionalized polymer may lower the rate of clearance from thecirculatory system via the reticuloendothelial system. For example, anon-functionalized polymer may balance an otherwise high concentrationof peptides, which can otherwise accelerate clearance by the subject,resulting in less delivery to the target cells.

The anti-NIH agent may be associated with the surface of, encapsulatedwithin, surrounded by, and/or dispersed throughout the therapeuticparticle. In another embodiment, the anti-NIH agent can be encapsulatedwithin the therapeutic particle.

Therapeutic compositions disclosed herein may, for example, be locallyadministered to a designated region of the blood vessel where the NIHoccurs. In still another embodiment, the therapeutic composition can beadministered via a medical device. In yet another embodiment, themedical device can be a drug eluding stent, needle catheter, or stentgraft. In one embodiment, the therapeutic compositions of this inventionpass through the endothelial layer of a blood vessel due to plaquedamage of the endothelial tissue and bind to collagen IV of the basementmembrane.

For example, contemplated particles may include CREKA bound to PEG(CREKA-PEG) (SEQ ID NO: 6), CREKA bound to PEG that is bound to a lipid(SEQ ID NO: 7) (e.g., CREKA-PEGDSPE (SEQ ID NO: 8)), and CREKA bound toPEG-PLGA (CREKA-PEG-PLGA (SEQ ID NO: 9)). Exemplary particles mayinclude a compound such as Formula VI and/or Formula VII:

wherein n is 20 to 1720; and

wherein R₇ is an alkyl group or H, R₈ is an ester or amide linkage,X+Y=20 to 1720, and Z=25 to 455. In other embodiments, X=0 to 1 molefraction Y=0 to 0.5 mole fraction.

In certain embodiments, the polymers of a disclosed particle may beconjugated to a lipid. The polymer may be, for example, alipid-terminated PEG. As described below, the lipid portion of thepolymer can be used for self assembly with another polymer, facilitatingthe formation of a particle. For example, a hydrophilic polymer could beconjugated to a lipid that will self assemble with a hydrophobicpolymer.

In some embodiments, lipids can be oils. In general, any oil known inthe art can be conjugated to the polymers used in the invention. In someembodiments, an oil may comprise one or more fatty acid groups or saltsthereof. In some embodiments, a fatty acid group may comprisedigestible, long chain (e.g., C₈-C₅₀), substituted or unsubstitutedhydrocarbons. In some embodiments, a fatty acid group may be a C₁₀-C₂₀fatty acid or salt thereof. In some embodiments, a fatty acid group maybe a C₁₅-C₂₀ fatty acid or salt thereof. In some embodiments, a fattyacid may be unsaturated. In some embodiments, a fatty acid group may bemonounsaturated. In some embodiments, a fatty acid group may bepolyunsaturated. In some embodiments, a double bond of an unsaturatedfatty acid group may be in the cis conformation. In some embodiments, adouble bond of an unsaturated fatty acid may be in the transconformation.

In some embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In another embodiment, a disclosed particle can be associated with(e.g., surrounded by) a small molecule amphiphilic compound e.g. havingas possible components: 1) a biodegradable polymeric material that formsthe core of the particle, which can carry bioactive drugs and releasethem at a sustained rate after cutaneous, subcutaneous, mucosal,intramuscular, ocular, systemic, oral or pulmonary administration; 2) asmall molecule amphiphilic compound that surrounds the polymericmaterial forming a shell for the particle; and 3) a targeting moleculethat can bind to a unique molecular signature on cells, tissues, ororgans of the body, such as the basement vascular membrane.

For example, a targeting molecule can be first chemically conjugated tothe hydrophilic region of a small molecule amphiphilic compound. Thisconjugate can be then mixed with a certain ratio of unconjugated smallmolecule amphiphilic compounds in an aqueous solution containing one ormore water-miscible solvents. In one embodiment, the targeting moleculecan be one or a plurality of peptides, small molecules, or combinationsthereof. The amphiphilic compound can be, but is not limited to, one ora plurality of the following: naturally derived lipids, surfactants, orsynthesized compounds with both hydrophilic and hydrophobic moieties.The water miscible solvent can be, but is not limited to: acetone,ethanol, methanol, and isopropyl alcohol. Separately, a biodegradablepolymeric material can be mixed with the agent or agents to beencapsulated in a water miscible or partially water miscible organicsolvent. In one embodiment, the biodegradable polymer can be any of thebiodegradable polymers disclosed herein, for example, poly(D,L-lacticacid), poly(D,L-glycolic acid), poly(ε-caprolactone), or theircopolymers at various molar ratios. The carried agent can be, but is notlimited to, one or a plurality of the following therapeutic agentsdiscussed below, including, for example, therapeutic drugs, imagingprobes, or hydrophobic or lipophobic molecules for medical use. Thewater miscible organic solvent can be but is not limited to: acetone,ethanol, methanol, or isopropyl alcohol. The partially water miscibleorganic solvent can be, but is not limited to: acetonitrile,tetrahydrofuran, ethyl acetate, isopropyl alcohol, isopropyl acetate, ordimethylformamide. The resulting polymer solution can then added to theaqueous solution of conjugated and unconjugated amphiphilic compound toyield particles by the rapid diffusion of the organic solvent into thewater and evaporation of the organic solvent.

Contemplated herein are particles that include surface modification,e.g. to enhance arterial uptake. Such surface modifying agents includefor example heparin, L-R-phosphatidylethanolamine, cyanoacrylate,epoxide, fibronectin, fibrinogen, ferritin, lipofectin,didodecyldimethylammonium bromide, and DEAEDextran, and any othersurface modifying agent disclosed in J Pharm Sci. 1998 October;87(10):1229-34, which is incorporated herein by reference in itentirety.

Polymer particles having more than one polymer or macromolecule present,and libraries involving such polymers or macromolecules are contemplatedherein. For example, in one set of embodiments, particles may containmore than one distinguishable polymers (e.g., copolymers, e.g., blockcopolymers), and the ratios of the two (or more) polymers may beindependently controlled, which allows for the control of properties ofthe particle. For instance, a first polymer may be a polymeric conjugatecomprising a targeting moiety and a biocompatible portion, and a secondpolymer may comprise a biocompatible portion but not contain thetargeting moiety, or the second polymer may contain a distinguishablebiocompatible portion from the first polymer. Control of the amounts ofthese polymers within the polymeric particle may thus be used to controlvarious physical, biological, or chemical properties of the particle,for instance, the size of the particle (e.g., by varying the molecularweights of one or both polymers), the surface charge (e.g., bycontrolling the ratios of the polymers if the polymers have differentcharges or terminal groups), the surface hydrophilicity (e.g., if thepolymers have different molecular weights and/or hydrophilicities), thesurface density of the targeting moiety (e.g., by controlling the ratiosof the two or more polymers), etc.

As a specific example, a particle may comprise a first polymercomprising a poly(ethylene glycol) and a targeting moiety conjugated tothe poly(ethylene glycol), and a second polymer comprising thepoly(ethylene glycol) but not the targeting moiety, or comprising boththe poly(ethylene glycol) and the targeting moiety, where thepoly(ethylene glycol) of the second polymer can have a different length(or number of repeat units) than the poly(ethylene glycol) of the firstpolymer. As another example, a particle may comprise a first polymercomprising a first biocompatible portion and a targeting moiety, and asecond polymer comprising a second biocompatible portion different fromthe first biocompatible portion (e.g., having a different composition, asubstantially different number of repeat units, etc.) and the targetingmoiety. As yet another example, a first polymer may comprise abiocompatible portion and a first targeting moiety, and a second polymermay comprise a biocompatible portion and a second targeting moietydifferent from the first targeting moiety.

In some cases, the particle can be a nanoparticle, i.e., the particlecan have a characteristic dimension of less than about 1 micrometer,where the characteristic dimension of a particle is the diameter of aperfect sphere having the same volume as the particle. For example, aparticle may have a characteristic dimension of the particle that may beless than about 300 nm, less than about 200 nm, less than about 150 nm,less than about 100 nm, less than about 50 nm, less than about 30 nm,less than about 10 nm, less than about 3 nm, or less than about 1 nm insome cases. In some embodiments, a disclosed particle may have adiameter of 50 nm-200 nm.

In general, the particles disclosed herein can be about 40 nm to about500 nm in size, for example, may be less than or equal to about 90 nm insize, e.g., about 40 nm to about 80 nm, e.g., about 40 nm to about 60nm. For example, particles less than about 90 nm in size, may reduceliver uptake by the subject, and may thereby allow longer circulation inthe bloodstream.

In an embodiment, particles disclosed herein may have a surface zetapotential ranging from about −80 mV to 50 mV. Zeta potential is ameasurement of surface potential of a particle. In some embodiments, theparticles can have a zeta potential ranging between 0 mV and −50 mV,e.g., between −1 mV and 50 mV. In some embodiments, the particles canhave a zeta potential ranging between −1 mV and −25 mV. In someembodiments, the particles can have a zeta potential ranging between−1.1 mV and −10 mV.

In other embodiments, the particles disclosed herein can includeliposomes, liposome polymer combinations, dendrimers, and albuminparticles that can be functionalized with a peptide ligand.

A polymeric conjugate to be used in the preparation of disclosedparticle may be formed using any suitable conjugation technique. Forinstance, two components such as a targeting moiety and a biocompatiblepolymer, a biocompatible polymer and a poly(ethylene glycol), etc., maybe conjugated together using techniques such as EDC-NHS chemistry(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride andN-hydroxysuccinimide) or a reaction involving a maleimide or acarboxylic acid, which can be conjugated to one end of a thiol, anamine, or a similarly functionalized polyether. In another set ofembodiments, a conjugation reaction may be performed by reacting apolymer that comprises a carboxylic acid functional group (e.g., apoly(ester ether) compound) with a polymer or other moiety (such as atargeting moiety) comprising an amine. For instance, a targeting moiety,such as a poly(amino-acid) ligand, may be reacted with an amine to forman amine-containing moiety, which can then be conjugated to thecarboxylic acid of the polymer. Such a reaction may occur as asingle-step reaction, i.e., the conjugation can be performed withoutusing intermediates such as N-hydroxysuccinimide or a maleimide.

For example, provided herein is a method of preparing therapeuticparticles, comprising: a) providing an anti-NIH agent; b) providing atleast one polymer; optionally c) providing a basement vascular membranetargeting peptide; d) mixing the at least one polymer with the anti-NIHagent to prepare particles; and optionally e) associating the particleswith the basement vascular membrane targeting peptide; such that thetherapeutic particles are formed. For example, at least one polymer canbe a copolymer of two or more polymers, such as PLGA and PEG. Alsoprovided herein is a method of preparing therapeutic particlescomprising: a) providing an anti-NIH agent; b) providing a firstpolymer; c) providing a second, non-functionalized polymer; optionallyd) providing a basement vascular membrane targeting peptide; e) reactingthe first polymer with the peptide to prepare a peptide-bound polymer;and f) mixing the peptide-bound polymer with the second,non-functionalized polymer and the anti-NIH agent; such that thetherapeutic particles are formed.

Disclosed particles and/or compositions may be delivered to a bloodvessel using a medical device such as a needle catheter, irrigationcatheter, balloon catheter, or can be delivered via intravenously e.g.,by i.v. infusion. In an exemplary embodiment, a balloon catheter, (e.g.the Genie™ balloon catheter available from Acrostak) and may be insertedinto a vessel having a lesion and in need of a stent. The balloon can beinflated to provide a pre-dilation of the vessel. The therapeuticcomposition can be, for example, then delivered to the blood vessel,followed by insertion of a bare metal stent, or the stent may be placedfirst and the composition then delivered.

Particles may be delivered to a subject in need thereof using deliverydevices that have been developed for endovascular local gene transfersuch as passive diffusion devices (e.g., double-occlusion balloon,spiral balloon), pressure-driven diffusion devices (e.g., microporousballoon, balloon-in-balloon devices, double-layer channeled perfusionballoon devices, infusion-sleeve catheters, hydrogel-coated balloons),and mechanically or electrically enhanced devices (e.g., needleinjection catheter, iontophoretic electric current-enhanced balloons,stent-based system), or any other delivery system disclosed in Radiology2003; 228:36-49, or Int J Nanomedicine 2007; 2(2):143-61, which areincorporated herein by reference in their entirety.

For example, as shown in FIG. 1A, a diagnostic/irrigation catheter 30 isused to deliver the therapeutic composition 40 to a blood vessel 10,such that the delivery of the therapeutic particles is in the blood flow20. In an exemplary different delivery method (FIG. 1B), a ballooncatheter 60 may be inserted into a blood vessel 10 with the blood flow20, proximal to the target delivery site. The balloon is inflated,preventing blood flow into area 50 of the blood vessel 10, then thetherapeutic composition 40 is injected into the catheter 60, thuslocalizing its delivery, with proximal landing of the particles.Alternatively, a diagnostic/irrigation catheter 30 may be used thatincludes balloon catheter 60 fed through it (FIG. 1C). Thediagnostic/irrigation catheter 30 is inserted into the blood vessel 10proximal to the target delivery site. The balloon catheter 60 is thenfed to a site 50 distal to the target delivery site. The balloon isinflated to prevent blood flow 20, then the therapeutic composition 40is introduced via the diagnostic/irrigation catheter, such that thedelivery is distal. An exemplary optional delivery method may include afirst balloon catheter fed through a second balloon catheter. The firstballoon catheter, proximal to the target delivery site, is inflated,followed by inflation of the second balloon catheter that has been fedto a position distal to the target delivery site. The therapeuticcomposition is introduced via the first balloon catheter such that it istrapped between the two balloons, e.g. includes both distal and proximaldelivery.

In some embodiments, disclosed compositions may be administeredintraveneously, e.g., systemically. For example, provided herein is amethod of preventing or deterring NIH in a blood vessel of a patientreceiving a bare metal stent in a lesion of said blood vessel,comprising intraveneously administering a composition that includestherapeutic particles, wherein said therapeutic particles comprise abasement vascular membrane targeting peptide and an anti-NIH agent. Suchtherapeutic particles may substantially localize in the blood vessele.g, that receives the bare metal stent. In some embodiments,intravenous administration may result in blood vessel localizationcomparable to or more substantially as compared to administration ofdisclosed compositions using e.g. a guide catheter and/or an angioplastyballoon.

As described above, disclosed compositions may provide a decreased orsubstantially comparable restenosis or NIH rate in the patient afterreceiving the vascular stent as compared to the restenosis rate obtainedby administration of a vascular stent, e.g. a bare metal stent alone.NIH or restenosis may be measured in angiographically (e.g, with binaryrestenosis), or clinically (e.g., target lesion revascularization).Binary restenosis, or angiographic restenosis, may include 50% or morediameter stenosis (DS) at follow up. It can be measured either by visualinspection or by quantitative coronary angiography (QCA). The percent ofbinary restenosis may correlate directly with lesion length; vesseldiameter; and/or the presence of diabetes. Target LesionRevascularization (TLR) usually includes a need for a repeatintervention at the site of the lesion due to the recurrence ofsymptoms. It is a clinical way to measure restenosis, although it canoccur for reasons other than restenosis, such as disease progression ora new lesion adjacent to the original treated area. Late loss studiesmay also be conducted and late loss may be independent of vessel size.Calculation of ate loss may allow the level of restenosis to beaccounted for in all vessels, regardless of size. Unlike binaryrestenosis, late loss does not allow a narrowed vessel of any magnitudeto go undetected. Late loss is measured in millimeters. The equation forthis process is: Minimum Lumen Diameter (MLD) Post Procedure−MLDFollow-up=Late Loss.

The present disclosure also provides pharmaceutical compositionscomprising particles as disclosed herein formulated together with one ormore pharmaceutically acceptable carriers. Exemplary materials which canserve as pharmaceutically acceptable carriers include, but are notlimited to, sugars such as lactose, glucose, and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients such as cocoabutter and suppository waxes; oils such as peanut oil, cottonseed oil;safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyllaurate;agar; detergents such as TWEEN™ 80; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator. Iffiltration or other terminal sterilization methods are not feasible, theformulations can be manufactured under aseptic conditions.

The pharmaceutical compositions of this invention can be administered toa patient by any means known in the art including oral and parenteralroutes, and/or systemically, e.g., by IV infusion or injection. In oneembodiment, the disclosed particles may be administered by IV infusion.In one embodiment, disclosed particles may be locally administered, forexample, brought into contact with the blood vessel wall or vasculartissue through a device.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can be used in the preparation of injectables.In one embodiment, the inventive conjugate is suspended in a carrierfluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v)TWEEN™ 80. The injectable formulations can be sterilized, for example,by filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Therapeutic particles disclosed herein may be formulated in dosage unitform for ease of administration and uniformity of dosage. The expression“dosage unit form” as used herein refers to a physically discrete unitof particle appropriate for the patient to be treated. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. For any particle, thetherapeutically effective dose can be estimated initially either in cellculture assays or in animal models, usually mice, rabbits, dogs, orpigs. The animal model can be also used to achieve a desirableconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Therapeutic efficacy and toxicity of particles can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose is therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose is lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionswhich exhibit large therapeutic indices may be useful in someembodiments. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosage for human use.

Also provided herein are kits that include a disclosed composition and astent, optionally with instructions for administering any of thecompositions described herein by any suitable technique as previouslydescribed, for example, orally, intravenously, pump or implantabledelivery device, or via another known route of drug delivery.

EXAMPLES Example 1 Simulation of Delivery of Therapeutic Particles

A balloon catheter is inserted into a length of 3.1 mm Tygon tubing.Water is injected at the opposite end at physiological blood pressure, 2psi (103 mmHg). This water is dyed blue in order to ascertain whetherthe balloon would withstand physiological pressure once inflated. Athree way valve with syrine is used for injection of a green solution toinflate the balloon and a second syringe to pull vacuum. A valve withsyringe attached is used for injection of a blue solution through alumen to distal end of balloon. After infusion of 0.3 mL of bluesolution, the pressure inside the tube is about 4 psi (206 mmHg), whichis higher than healthy human blood pressure, and the balloon withstandspressure with no leakage downstream.

Example 2 Tissue Binding and Persistence of Nanoparticle Formulation inDenuded Rabbit Iliac Arteries

16 New Zealand white rabbits were used. Animals were kept in accordancewith Institutional Animal Care and Use Committee (IACUC) protocols. Toprevent or reduce the occurrence of thrombotic events, animals weretreated with acetylsalicylic acid (40 mg, per os [PO]) daily, at leastone day prior to the beginning of the study. Animals were anesthetizedaccording to the testing facility standard operating procedures. Afterinduction of anesthesia, the left or right carotid artery was accessedwith an incision made in the throat region. An arterial sheath wasintroduced and advanced into the artery. As an anti-coagulation therapy,an initial bolus of heparin (˜70 IU/kg) was given following cannulationof the carotid artery.

Before the first angiogram, 1 mL of nitroglycerine (0.5 mg/mL) IV wasgiven. The iliac artery was circumscribed (from the femoral to theinternal iliac branch) and Quantitative Angiography (QA) was performedto document the vessel size. Then, balloon injury was performed in bothiliac arteries of each rabbit. The appropriate balloon (balloon toartery ratio of 1.3:1 or more if needed) was advanced over the guidewireto traverse the distal portion of the pre-selected injury site. Theinflated balloon was then retracted from the femoral artery back intothe aorta to enable denudation of the target vasculature. The balloonwas deflated and re-advanced to traverse the target injury site. Theballoon was deflated while it was in the terminal descending aorta andthe denudation procedure repeated one more time (total of 3 times). Apost-denudation angiogram was performed and TIMI flow was assessed. Ananimal with post-TIMI flow of 0 or 1 received an intra-arterial infusionof nitroglycerine at the discretion of the interventionalist to restorethe flow to 2 or 3. The guide wire was then advanced to the oppositeiliac artery, and the injury procedure was repeated with the appropriatesized balloon for the second artery.

PLA-PEG-lipid-CREKA (SEQ ID NO: 1) nanoparticles with a diameter of ˜240nm and labeled with a fluorescent tag were administered with a particledye load of about 1 wt. %. The targeted nanoparticles had aconcentration of 6 mg/ml in the administered composition before 50%dilution with contrast media. Animals were dosed with 3 mL of 6 mg/mLnanoparticles in 50% contrast media such that for local delivery, 3 mLof solution were delivered to each iliac artery (resulting in 18 mg doseof nanoparticles delivered to all animals).

Four methods were used for delivery of the nanoparticles: guidecatheter, angioplasty balloon, guide catheter and angioplasty balloon,and systemic i.v. Four animals received the nanoparticle solution byeach of the four methods. For each method, two animals were sacrificedwithin 30 minutes of delivery (“acute”) and the other two animals weresacrificed 24 hours after delivery. In acute cases of local delivery,5-15 minutes elapsed between injury and delivery, and 5 minutes elapsedbetween delivery and sacrifice. For acute i.v. administration, an hourelapsed between delivery and sacrifice.

Guide catheter: After both arteries had been balloon denuded, the guidecatheter was advanced over the guidewire near the delivery site. Twomilliliters of the nanoparticle formulation combined with an appropriateamount of contrast agent (1 mL) for a total delivery volume of 3 mL wasadministered in one minute via the guide catheter locally to the site ofballoon injury. This procedure was repeated for the other injured iliacartery.

Angioplasty balloon: After both arteries had been balloon denuded, theguidewire was removed. The balloon was then inflated sufficiently toocclude blood flow in the iliac artery. Two milliliters of thenanoparticle formulation mixed with an appropriate amount of contrastagent (1 mL) for a total delivery volume of 3 mL was administered viathe lumen of the balloon catheter while the artery was occluded over aperiod of one minute. The balloon was then deflated, retracted, andadvanced over the guidewire to the other iliac artery. The deliveryprocedure was repeated in the second artery.

Guide catheter with angioplasty balloon: After both arteries had beenballoon denuded, a 5F guide catheter was advanced to the first iliacartery. An angioplasty balloon was then advanced through the guidecatheter to the delivery site, approximately 1-2 cm distally from thetip of the guide catheter. The balloon was inflated sufficiently toocclude blood flow in the iliac artery. Two milliliters of thenanoparticle formulation mixed with an appropriate amount of contrastagent (1 mL) for a total delivery volume of 3 mL was then administeredvia the guide catheter while the artery was occluded over a period ofapproximately one minute. The balloon was then deflated, and both theballoon and guide catheter were retracted, and advanced to the otheriliac artery. The delivery procedure was repeated in the second artery.

Systemic i.v.: Immediately after balloon denudation, the animalsreceived a single i.v. injection of 2 mL of nanoparticle formulationcombined with 1 mL of saline for a total delivery volume of 3 mL, in thesame volume ratio as the nanoparticle formulations with contrast for theother delivery methods. Injection was at the marginal vein of the ear.

Upon completion of nanoparticle delivery to the second iliac artery inanimals from the acute cohort, with the exception of the systemic i.v.delivery group, the animals were kept deeply anesthetised beforeeuthanasia with a rapid bolus of pentobarbital. This bolus wasadministered within 30 minutes after delivery.

Animals from the 24 hour cohort, as well as from the systemic i.v.delivery group, were allowed to regain consciousness. At the appropriatesacrifice time (1 hour after nanoparticle delivery for the acutesystemic i.v. group, or 24 hours after nanoparticle delivery for all the24 hour groups), the animals were first tranquilized with acepromazineadministered sub-cutaneously [SC]. Heparin (˜70 IU/kg) was administeredprior to sacrifice for the 24 hour groups only. The animals were theneuthanized with a rapid bolus of pentobarbital.

After euthanasia, the soft tissue surrounding the external iliacarteries was dissected off gently to expose the external artery wall,from about 1 cm proximal to the internal iliac branch to about 1 cmdistal to the femoral branch. Between these two branches, a central 2 cmlong segment was delimited (in the delivery region), using a dot ofblack ink at the proximal end and a dot of red ink at the distal end.The ink was allowed to dry, then the artery was explanted, by cutting itfrom about 1 cm proximal to the internal iliac branch to about 1 cmdistal to the femoral branch. The explanted segment was gently rinsed byimmersion in approximately 10 milliliters of physiologic saline. Excesssaline was gently removed from the explanted iliac vessels by gentlepadding on absorbent paper.

The explanted segments were then sectioned and embedded in an OCT(optimal cutting temperature) cryomold. After freezing in liquidnitrogen, the arteries were cut into 10 sections each, resulting in 320sections from 16 animals having 2 iliac arteries each. The sections weremounted on slides and viewed at 10× magnification for lumen detail and4× magnification for the whole section. The segments were scored on ascale of 0-3 for the amount of fluorescence observed above tissuebackground, with 0 being no fluorescence, 1 very few points offluorescence, 2 many points of fluorescence, and 3 being a continuouslayer of fluorescence. A total of 1800 images were collected and 1630were scored. Table 1 provides the average score for each delivery methodat the acute and 24 hour time points.

TABLE 1 Average Score - Average Score - Delivery Acute - 24 hour -Delivery Mode Location Animals 1 and 2 Animals 1 and 2 Guide CatheterLocal 1.8 0.08 2.1 0.00 Angioplasty Local 1.9 0.09 Balloon 1.7 0.04Guide Catheter Local 1.1 0.13 with Angioplasty 2.2 0.12 Balloon i.v.Systemic 2.5 0.07 2.2 0.02

Of the images, 33 were identified as having good fluorescence results.Twenty of these images were from the systemic i.v. group, 6 were fromthe guide catheter group, 4 were from the guide catheter withangioplasty balloon group, and 3 were from the angioplasty balloongroup. These results illustrate the effectiveness of systemic i.v.delivery to target nanoparticles of the invention to a site of arterialinjury.

Example 3 General Synthetic Procedure for Particles

Product constituents were dissolved in organic solvent system (generally79% ethyl acetate, 21% benzyl alcohol) at a given solids concentration(generally 15% w/w). This organic phase was emulsified with a rotorstator homogenizer with aqueous phase pre-saturated with solvent(generally 2% benzyl alcohol, 4% ethyl acetate, and sometimes withsodium cholate included as a surfactant). The weight ratio ofaqueous:organic was commonly 10:1. This emulsion was formed into a fineemulsion though high pressure homogenization on a microfluidizer(generally microfluidics 110S air driven homogenizer, processingpressure ˜9000 psi). The emulsion was quenched into a cold water quench,generally at 10:1 quench:emulsion ratio. Polysorbate-20 (T-20) was addedto the quench to solubilize unencapsulated drug. The slurry was thenprocessed with ultrafiltration/diafiltration to remove T-20 andunencapsulated drug. Solids and drug assays were performed on the finalslurry to determine drug loading. In order to provide freeze-thawstability, the final slurries were brought to 10% sucrose (w/w) thenstored frozen.

Example 4

Particles with placebo containing ˜10 kDa PLA and 5% DSPE-PEG5k wereprepared using the procedure of Example 3. No surfactant was used andthe particle size after 3 passes on the homogenizer was 153.5 nm.

Example 5 Effect of Lipid Content on Particle Size

DSPE-PEG5k at 1%, 5%, and 10% of solids were prepared as in Example 2using PLA ˜10 kDa and the effect on particle size was investigated.Additional PEG-lipid decreased the particle size, as shown in FIG. 2.

Shorter 2 kDa PEG chains on the lipid, DSPE-PEG2k, were alsoinvestigated for effect on particle size using DSPE-PEG2k at 1%, 5%, and10% of solids. The PLA used was ˜10 kDa. The particle size, shown inFIG. 3, was reduced relative to the above, which used DSPE-PEG5k. Thismay be because the shorter PEG chain may give the PEG-lipid bettersurfactant qualities. Further, the shorter PEG chain may mean that at agiven wt % PEG-lipid, there is a greater number of PEG-lipids presentwhen the PEG chain is shorter.

Example 6 Paclitaxel Release

Particles with PTXL (paclitaxel) incorporated at a target load of 20%,with 5% DSPE-PEG5k were prepared as in Example 2 (Lot 126) The PLA usedwas ˜10 kDa. The final particle size was 174.7 nm, and the PTXL load was16.1%.

Lots with PTXL incorporated at a target load of 20% were also preparedwith 5% (Lot 135A) or 10% (135B) DSPE-PEG2k. The PLA used was ˜10 kDa.The final particle size was 133.2 nm (A) and 94.2 nm (B), and the PTXLload was 15.1% (A) and 13.1% (B). This indicates good encapsulation isstill achieved, even with particles under 100 nm. Release testing at 37°C. on these particles is shown in FIG. 4.

Example 7 Paclitaxel Release

PTXL was incorporated into particles containing 16.5/5 PLA/PEG copolymerusing the procedure of Example 3. The final particle size was 85 nm andthe PTXL load was 6.6%. The particle size was slightly smaller than thetarget of ˜100 nm (Lot 148)

Lot 152 was prepared by incorporating PTXL at a target load of 20%, with5% DSPE-PEG5k, and high MW PLA, ˜85 kDa. The final particle size was242.8 nm, and the PTXL load was 17.2%.

Lot 156(A) was prepared by incorporating PTXL at a target load of 20%,with 5% DSPE-PEG2k, using PLA with ˜22 kDa. The particle size was 133.9nm and 15.2% drug load.

Lot 156(A) was prepared by incorporating PTXL at a target load of 20%,with no PEG-lipid, using ˜10 kDa PLA.

The release testing, shown in FIG. 5, indicates that the cause of thefast release appears to be the low MW (˜10 kDa) PLA. High MW (˜85 kDa)PLA exhibited significantly slower release. This suggests that therelease can be altered through the use of higher MW PLAs. It is possiblethe large particle size played a role in decreasing the release, butprevious work has shown little dependence of release on particle size.

Example 8 Pacitaxel Release

Lot 41-191 incorporated PTXL at a target load of 20%, with 10%DSPE-PEG2k, 20% Alexa fluor 647-PLA, and ˜22 kDa PLA. The final particlesize was 135.2 nm, and the PTXL load was 16.8%. This is the non-targetedmaterial for CV-INVIV-003. Alexa fluor 647-PLA is incorporated so thatarteries can be assessed with fluorescence microscopy to assess particlebinding.

For lot 41-205, PTXL was incorporated at a target load of 20%, with ˜10%DSPE-PEG2k, 20% Alexa fluor 647-PLA, ˜22 kDa PLA, and Smol % of eitherDSPE-PEG2k-CREKA (SEQ ID NO: 12) (41-205(A)) or DSPE-PEG2k-CARYLQKLN(SEQ ID NO: 13) (41-205(B)). The final particle size was 122.8 nm (A)and 145.1 nm (B), and the PTXL load was 14.9% (A) and 16.2% (B). Theseare the targeted PEG-lipid formulations in CV-INVIV-003.

Lot 61-1 incorporated PTXL at a target load of 20%, with 20% Alexa fluor647-PLA, 10 mol % PLA-PEG-CREKA, with 16.5/5 PLA/PEG. The final particlesize was 103.5 nm, and the PTXL load was 14.2%. This is the copolymertargeted material for CV-INVIV-003. Release profiles are shown in FIG.6.

Example 9 Animal Model—Dosing with Paciltaxel

Rabbits were used as in Example 2. One femoral artery in anesthetizedrabbits was denuded with a balloon injury model. Briefly, a ballooncatheter was overinflated ˜30% then pulled along the artery three times,intended to injury the artery and effectively remove the endothelialcells. The animals were then dosed with a solution of a givenformulation, at 1 mg/kg PTXL, including abraxane. Abraxane is PTXL in analbumin nanoparticulate formulation, which rapidly dissolved uponadministration, mimicking free drug.

Blood was drawn from the animal at 20, 40 and 60 minutes, at which timethey were sacrificed. As the release profiles of all formulations werevery similar, any significant differences in the plasma profiles likelyindicate altered particle circulation times/clearance. The data shown inFIG. 7 indicates that the longest circulating particles are those of thecopolymer, while the lipid particle formulations appear to have a fasterclearance rate. However all formulations do successfully exhibitprolonged circulation times, as free drug is rapidly cleared from theplasma, as modeled in the abraxane arms.

Example 10 Paciltaxel Dosing Via IV in Animal Model

Rabbits were used as in Example 2 Paclitaxel loaded particles wereadministered to rabbit injured vessels via IV injection. Each animal hadone iliac artery injured via balloon expansion while the other arterywas undisturbed. Nanoparticles were injected in the marginal ear veinimmediately after injury and blood drawn every 20 minutes to monitorpaclitaxel blood levels. The animals were sacrificed after 60 minutesand vessels removed to measure their Paclitaxel content.

Multiple particles were tested including non-targeted lipid basedparticles, targeted lipid based particles, and targeted copolymer basedparticles. Table 2 lists the formulation details for the particlestested. Ligands were either attached to the DSPE-PEG lipids or PLA-PEGcopolymers, as described above, depending on the formulation.

TABLE 2 Formulations for animal study PTXL Particle Nano- TagetingLoading Size particle ligand Payload (%) (nm) 1 lipid CREKA (SEQ PTXL 15 123 ID NO: 1) 2 lipid CARYLQKLN PTXL 16  145 (SEQ ID NO: 2) 3 lipidnone PTXL 17  135 4 copolymer CREKA (SEQ PTXL 14  104 ID NO: 1) 5 N/AN/A Albumin- 10 ~130 bound paclitaxel

Release rate from formulations 1 through 4 are shown in FIG. 8. Thenanoparticles had similar release rates in an in vitro release assaywhich should allow comparisons between the different formulations in theanimals. Blood levels are shown in FIG. 9. The copolymer (formulation 4)had sustained blood concentration compared to the albumin paclitaxel andlipid based nanoparticles.

FIG. 10 indicates vessel levels. The copolymer had significantly highertissue levels than other formulations.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

1. A method of preventing or deterring NIH in a blood vessel of apatient receiving a bare metal stent in a lesion of said blood vessel,comprising: administering a composition comprising therapeuticparticles, wherein said therapeutic particles comprise: about 1 to about20 mole percent PLA-PEG-basement vascular membrane targeting peptide,wherein the targeting peptide comprises PLA having a number averagemolecular weight of about 15 to about 20 kDa and PEG having a numberaverage molecular weight of about 4 to about 6 kDa; about 10 to about 25weight percent anti-neotimal hyperplasia (NIH) agent; about 50 to about90 weight percent non-targeted poly-lactic acid-PEG, wherein thetherapeutic particles are capable of releasing the anti-NIH agent to abasement vascular membrane of a blood vessel for at least about 8 hourswhen the therapeutic particles are placed in the blood vessel.
 2. Themethod of claim 1, wherein the method provides a decreased orsubstantially comparable NIH rate at about 2 weeks after receiving saidbare metal stent as compared to a patient receiving a stent comprisingan anti-NIH agent at about 2 weeks.
 3. The method of claim 1, whereinthe composition is administered using a catheter.
 4. The method of claim1, wherein the composition is administered intravenously.
 5. The methodof claim 1, wherein the composition is administered substantiallysimultaneously when the patient receives the stent.
 6. The method ofclaim 1, wherein the composition is administered before the patientreceives the stent.
 7. The method of claim 6, wherein the composition isadministered before a balloon catheter is introduced into the bloodvessel.
 8. The method of claim 6, wherein the composition isadministered after a balloon catheter is introduced into the bloodvessel.
 9. The method of claim 1, wherein the basement vascular membranetargeting peptide comprises a sequence selected from the groupconsisting of AKERC (SEQ ID NO: 3), CREKA (SEQ ID NO: 1), ARYLQKLN (SEQID NO: 4), and AXYLZZLN (SEQ ID NO: 5), wherein X and Z are variableamino acids.
 10. The method of claim 9, wherein the basement vascularmembrane targeting peptide comprises a sequence selected from the groupconsisting of AKERC (SEQ ID NO: 3) and CREKA (SEQ ID NO: 1).
 11. Themethod of claim 1, wherein the basement vascular membrane targetingpeptide comprises a sequence selected from the group consisting of CREKA(SEQ ID NO: 1) and CARLYQKLN (SEQ ID NO: 2).
 12. The method of claim 1,wherein the non-targeted poly-lactic acid-PEG has PLA with a numberaverage molecular weight of about 20 kDa to about 25 kDa.
 13. The methodof claim 1, wherein the anti-neointimal hyperplasia (NIH) agent ispaclitaxel.